Bacterial resistance to antimicrobials is among the biggest challenges affecting human health. Many resistance mechanisms developed by bacteria involve adaptation of cell wall components that lead to decreased interaction with, or permeability to antimicrobial compounds. One such mechanism uses amino acids (aa), carried by tRNAs, to aminoacylate phosphatidylglycerol (PG), one of the main constituents of the bacterial membrane. Since the initial discovery of this pathway, studies have shown that lipid aminoacylation enzymes (more generally referred to as aminoacyl-phosphatidylglycerol synthases; aaPGSs) are surprisingly diverse, and have several aa in their repertoire for altering the chemical makeup of bacterial membranes. Numerous studies have demonstrated that PG aminoacylation enhances bacterial resistance to antibiotics that target the membrane (such as cationic antimicrobial peptides, CAMPs), as well as the virulence of pathogens from multiple phyla (i.e., Firmicutes and Proteobacteria). However, lipid modification systems have been largely ignored in the Actinobacteria, an important bacterial phylum that includes pathogens of major importance to human health, such as the etiological agents of tuberculosis, diphtheria, nocardiosis, and actinomycosis. An extensive genome analysis showed that bacteria in this phylum frequently harbor multiple (up to six) aaPGS homologs, suggesting that Actinobacteria display a high level of functional diversity in their lipid modification systems. Our preliminary studies validated this hypothesis, leading to discovery of several novel lipid modifications systems in corynebacteria that are important for antimicrobial resistance and virulence. A multitude of other lipid-modifying enzymes in Actinobacteria are yet to be described, and we hypothesize that some of these proteins support novel functions as well. These studies are important because aaPGSs represent general factors for membrane adaptation and are expressed in species of extreme importance to human health. Identification of novel lipid modifications will not only increase our understanding of fundamental bacterial defense mechanisms, but will reveal new targets for development of antimicrobial compounds. Our long-term goal is to define the repertoire of lipid modifications across bacterial species and to characterize their role in cellular physiology, antimicrobial resistance, and pathogenesis. The aims of this proposal, which will lay the groundwork for future studies, are to i) explore aaPGS homolog diversity in Actinobacteria and characterize the biochemical functions of representative enzymes from human pathogens in the genera Mycobacterium, Nocardia, Rhodococcus, and Streptomyces; and ii) determine the role of these proteins in resistance to CAMPs of the human immune system and other antimicrobial agents.